Tacrolimus in human whole blood was quantified utilizing a simple, efficient, and reproducible approach. With Phenomenex, Strata-X SPE cartridges, Tacrolimus and internal standard Tacrolimus 13CD4 were extracted from human whole blood using a solid phase extraction technique. Chromatographic separation was performed using an ACE CN (4.6 x 75mm) 1.5μm column as the stationary phase, while a mobile phase made up of acetonitrile and ammonium acetate (90:10 v/v) flowed at a rate of 0.60 mL/min followed by MRM. Electrospray ionization was used to monitor the eluents with Tacrolimus set at transition m/z 821.4→ 768.5 for Tacrolimus and m/z 826.43→ 773.27 for Tacrolimus (IS), respectively. Tacrolimus and its internal standard had a retention period of 3.25 minutes. In accordance with US-FDA guidelines, the technique was validated for precision, accuracy, specificity, recovery, matrix effect, linearity, and limit of detection. Tacrolimus quantification was carried out with a precise and accurate approach, as shown by the assay validation findings, which all fulfilled the necessary acceptance criteria. The range of the validated test was 0.204 to 60.080 ng/mL. No matrix impact, carryover, or interference were noticed. The calibration curve was confirmed to be linear, with a r2 value of 0.99. Tacrolimus was found to have a LOQ signal-to-noise (S/N) ratio of 23.789. The devised technique demonstrated high analyte and IS recovery. When the concentration of Tacrolimus in the matrix was evaluated for stability, no discernible change was found. The bioanalytical method validated in this research is suitable and practicable for quantitating Tacrolimus in human whole blood samples obtained using LC-MS/MS.

Human whole blood, LC-MS/MS, Method development, Method validation, Tacrolimus